DESCRIPTION: Changes in iron homeostasis have dramatic consequences for human health. Cancer, heart disease, neural disorders - even aging itself - have been linked to excess iron or iron-induced oxidative damage. Storage of ferrous (Fe(2+) iron in a non-toxic (Fe(3+) mineral oxide form (FeOOH) by ferritin (Ft), a spherical, hollow protein, thus assumes tremendous significance in human physiology. Organ-specific modulation of the rate of iron uptake by Ft, a long-term goal with enormously favorable biomedical implications, presupposes a basic understanding of the uptake mechanism. Unfortunately, several aspects of iron traversal across the 20-A thick protein sheath are not completely understood. These include the iron coordination environment during oxidation, transport, and mineralization, as well as mechanistic differences between protein L and H subunits. Detailed information about the iron coordination sphere at (and within) the protein surface over the course of uptake and biomineralization will be generated using surface enhanced Raman scattering (SERS), a vibrational spectroscopy with extraordinary sensitivity for molecules in proximity to roughened Ag surfaces. The use of Raman spectroscopy to provide detailed molecular-level information about non-heme iron binding sites in proteins is extremely well-documented, as are numerous studies demonstrating retention of Ft quaternary structure when adsorbed to metal surfaces. Accordingly, preliminary SERS studies at two different excitation wavelengths of well-defined Fe:Ft complexes (0.5, 2,4, and 6 Fe: subunit) adsorbed to SERS-active colloidal Ag surfaces reveal dramatic spectral differences that clearly emanate from distinct iron-containing species. Detailed information of this nature on stable intermediates in the biomineralization of iron by ferritin has never before been available at physiological temperatures. The proposed work will expand on these exciting results to generate a step-by-step molecular view of the early stages of iron uptake by Ft, with a particular focus on elucidating the amino acids involved in oxidation and transport. Experiments on well-defined Ft samples from several organisms, with varying H/L ratios, different ligand sets (i.e. site-directed mutants), and different Fe:Ft ratios will be studied as a function of excitation wavelength, temperature, and iron oxidant (inner sphere vs. outer sphere). This research will not only greatly improve understanding of iron homeostasis but demonstrate the utility of SERS as a powerful probe of metalloprotein structure and function.